Ex vivo imaging and analysis of ROS generation correlated with microglial activation in rat model with acute neuroinflammation induced by intrastriatal injection of LPS

Background: Amyotrophic lateral sclerosis (ALS) is a neurological disorder clinically characterized by motor system dysfunction, with intraneuronal accumulation of the TAR DNAbinding protein 43 (TDP-43) being a pathological hallmark. Riluzole is a primarily prescribed medicine for ALS patients, while its therapeutical efficacy appears limited. TDP-43 transgenic mice are existing animal models for mechanistic/translational research into ALS. Methods: We developed a transgenic rat model of ALS expressing a mutant human TDP-43 transgene (TDP-43M337V) and evaluated the therapeutic effect of Riluzole on this model. Relative to control, rats with TDP-43M337V expression promoted by the neurofilament heavy subunit (NEF) gene or specifically in motor neurons promoted by the choline acetyltransferase (ChAT) gene showed progressive worsening of mobility and grip strength, along with loss of motor neurons, microglial activation, and intraneuronal accumulation of TDP-43 and ubiquitin aggregations in the spinal cord. Results: Compared to vehicle control, intragastric administration of Riluzole (30 mg/kg/d) did not mitigate the behavioral deficits nor alter the neuropathologies in the transgenics. Conclusion: These findings indicate that transgenic rats recapitulate the basic neurological and neuropathological characteristics of human ALS, while Riluzole treatment can not halt the development of the behavioral and histopathological phenotypes in this new transgenic rodent model of ALS.

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Vitamin D Deficiency Induces Chronic Pain and Microglial Phenotypic Changes in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22073604 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3604

Author(s):

Nicola Alessio ◽

Carmela Belardo ◽

Maria Consiglia Trotta ◽

Salvatore Paino ◽

Serena Boccella ◽

...

Keyword(s):

Spinal Cord ◽

Vitamin D ◽

Chronic Pain ◽

Vitamin D Deficiency ◽

Morphological Analysis ◽

Pain Perception ◽

Ex Vivo ◽

Ros Generation ◽

Peroxisome Proliferator ◽

Neuroprotective Effects

The bioactive form of vitamin .D, 1,25-dihydroxyvitamin D (1,25D3), exerts immunomodulatory actions resulting in neuroprotective effects potentially useful against neurodegenerative and autoimmune diseases. In fact, vitamin D deficiency status has been correlated with painful manifestations associated with different pathological conditions. In this study, we have investigated the effects of vitamin D deficiency on microglia cells, as they represent the main immune cells responsible for early defense at central nervous system (CNS), including chronic pain states. For this purpose, we have employed a model of low vitamin D intake during gestation to evaluate possible changes in primary microglia cells obtained from postnatal day(P)2-3 pups. Afterwards, pain measurement and microglia morphological analysis in the spinal cord level and in brain regions involved in the integration of pain perception were performed in the parents subjected to vitamin D restriction. In cultured microglia, we detected a reactive—activated and proliferative—phenotype associated with intracellular reactive oxygen species (ROS) generation. Oxidative stress was closely correlated with the extent of DNA damage and increased β-galactosidase (B-gal) activity. Interestingly, the incubation with 25D3 or 1,25D3 or palmitoylethanolamide, an endogenous ligand of peroxisome proliferator-activated-receptor-alpha (PPAR-α), reduced most of these effects. Morphological analysis of ex-vivo microglia obtained from vitamin-D-deficient adult mice revealed an increased number of activated microglia in the spinal cord, while in the brain microglia appeared in a dystrophic phenotype. Remarkably, activated (spinal) or dystrophic (brain) microglia were detected in a prominent manner in females. Our data indicate that vitamin D deficiency produces profound modifications in microglia, suggesting a possible role of these cells in the sensorial dysfunctions associated with hypovitaminosis D.

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AB0069 LORECIVIVINT (SM04690), AN INTRA-ARTICULAR, SMALL-MOLECULE CLK/DYRK1A INHIBITOR THAT MODULATES THE WNT PATHWAY, AS A POTENTIAL TREATMENT FOR MENISCAL INJURIES

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.6454 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1335.2-1335

Author(s):

T. Seo ◽

V. Deshmukh ◽

Y. Yazici

Keyword(s):

Small Molecule ◽

Rat Model ◽

Wnt Pathway ◽

Ex Vivo ◽

Meniscal Tear ◽

Knee Oa ◽

Meniscus Degeneration ◽

Anti Inflammatory ◽

Meniscal Damage

Background:Meniscal injuries, associated with pain, stiffness, and localized swelling, are the most common pathology of the knee with a prevalence of 61 per 100,000.1Meniscal damage is a frequent finding on MRI images of knee osteoarthritis (OA)2; while a meniscal tear can lead to knee OA, knee OA can also lead to a spontaneous meniscal tear.3Efforts to repair meniscal damage have been largely unsuccessful and do not prevent the progression of degenerative changes that lead to knee OA.4The Wnt signaling pathway has been shown to be regulated during meniscal development,5,6suggesting that manipulation of this pathway may influence the regenerative capacity of the meniscus. Lorecivivint (LOR; SM04690) is an intra-articular (IA), small-molecule CLK/DYRK1A inhibitor that modulates the Wnt pathway.Objectives:LOR was evaluated in preclinical studies to determine its protective and anabolic effects in ex vivo explants and in a rat model of chemically induced inflammatory meniscus degeneration.Methods:Effects of LOR (30 nM) on expression of matrix metalloproteinases (MMPs) in cultured rat menisci treated with IL-1B were measured by qPCR. In vivo, LOR activity was evaluated in a rat model of monosodium iodoacetate (MIA) injection-induced inflammatory meniscus degeneration. A single IA injection of MIA was immediately followed by a single IA injection of LOR (0.3 ug) or vehicle. Knees were harvested on Days 1, 4, and 11 and menisci were isolated. Anti-inflammatory effects were evaluated by measuringTNFAandIL6expression by qPCR. Meniscus protection was evaluated by qPCR for MMPs and aggrecanase and anabolic effects by qPCR for collagens.Results:In ex vivo meniscal explants, LOR inhibited expression ofMMP1,MMP3, andMMP13compared to DMSO (P<0.01). In vivo, LOR significantly decreased expression of these MMPs and aggrecanase (P<0.05) compared to vehicle in the rat model of inflammatory meniscus degeneration at Day 4 after MIA injection. In addition, LOR reduced expression of inflammatory cytokinesTNFAandIL6at Day 4 compared to vehicle. Finally, LOR increased expression of collagen types I, II, and III at Day 11 after MIA injection.Conclusion:LOR exhibited protective effects in the meniscus ex vivo and in vivo by reducing the expression of catabolic enzymes compared to control. Anti-inflammatory effects of LOR were demonstrated by inhibition of inflammatory cytokine expression. Compared to vehicle, LOR increased expression of collagens in vivo, indicating potential meniscal anabolic effects. These data support further investigation of LOR as a potential disease-modifying therapy for meniscal injuries.References:[1]Logerstedt D and Snyder-Mackler L.J Orthop Sports Phys Ther. 2010[2]Englund M, et al.Rheum Dis Clin North Am. 2009[3]Englund M, et al.Radiol Clin North Am. 2009[4]von Lewinski, et al.Knee Surg Sports Traumatol Arthrosc. 2007[5]Pazin DE, et al.ORS 2012 Annual Meeting. Paper No. 0221[6]Pazin DE, et al.Dev Dyn. 2012Disclosure of Interests:Tim Seo Shareholder of: Samumed, LLC, Employee of: Samumed, LLC, Vishal Deshmukh Shareholder of: Samumed, LLC, Employee of: Samumed, LLC, Yusuf Yazici Shareholder of: Samumed, LLC, Grant/research support from: Bristol-Myers Squibb, Celgene, and Genentech, Consultant of: Celgene and Sanofi, Employee of: Samumed, LLC

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Inhibition of ionotropic GluR signaling preserves oligodendrocyte lineage and myelination in an ex vivo rat model of white matter ischemic injury

Acta Neurobiologiae Experimentalis ◽

10.21307/ane-2021-022 ◽

2021 ◽

Vol 81 (3) ◽

pp. 233-248

Author(s):

Mohamed A. Al-Griw ◽

Michael G. Salter ◽

Ian C. Wood

Keyword(s):

White Matter ◽

Rat Model ◽

Ex Vivo ◽

Ischemic Injury

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Effects of intracavernous administration of adrenomedullin on erectile function in in/ex vivo rat model

The Japanese Journal of Urology ◽

10.5980/jpnjurol.92.288_2 ◽

2001 ◽

Vol 92 (2) ◽

pp. 288

Keyword(s):

Rat Model ◽

Erectile Function ◽

Ex Vivo

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ILEX PARAGUARIENSIS, EXERCISE AND CARDIOPROTECTION: A retrospective analysis

10.1101/452946 ◽

2018 ◽

Author(s):

Fábio Cahuê ◽

José Hamilton Matheus Nascimento ◽

Luciane Barcellos ◽

Veronica P. Salerno

Keyword(s):

Data Science ◽

Molecular Mechanisms ◽

Cardiac Tissue ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Ilex Paraguariensis ◽

Ros Generation ◽

Yerba Mate ◽

Reductive Stress ◽

Apparent Loss

AbstractStudies on strategies to generate cardioprotective effects have been on the rise. Previous work by our group with an ex vivo model of ischemia/reperfusion has shown that both the short-term consumption of yerba mate and exercise can each induce protection of cardiac function independently. Surprising, the two strategies together do not, with an apparent loss of their respective cardioprotection activity. To improve our understanding of the mechanisms involved without reperforming the experiments, we have conducted a retrospective data science-analysis that have produced new insights. The analysis shows that yerba mate generated reductive stress. Alone, this stress increased redox damage in the heart that appears to have led to a protective conditioning. In combination with exercise, the effects of mate inhibited the intermittent ROS generation promoted by exercise alone, which diminished the adaptive response in the heart. These results suggest that an understanding of the molecular mechanisms involved with the yerba mate-promoted reductive stress in cardiac tissue could lead to improved strategies to induce cardioprotection.

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Detecting Validated Intracellular ROS Generation with 18F-dihydroethidine-Based PET

Molecular Imaging and Biology ◽

10.1007/s11307-021-01683-0 ◽

2021 ◽

Author(s):

Edward C. T. Waters ◽

Friedrich Baark ◽

Zilin Yu ◽

Filipa Mota ◽

Thomas R. Eykyn ◽

...

Keyword(s):

Oxidative Stress ◽

Contractile Function ◽

Ex Vivo ◽

Glutathione Depletion ◽

Ros Generation ◽

Cardiac Contractile ◽

Rat Hearts ◽

Intracellular Ros ◽

Cardiac Contractile Dysfunction

Abstract Purpose To determine the sensitivity of the 18F-radiolabelled dihydroethidine analogue ([18F]DHE) to ROS in a validated ex vivo model of tissue oxidative stress. Procedures The sensitivity of [18F]DHE to various ROS-generating systems was first established in vitro. Then, isolated rat hearts were perfused under constant flow, with contractile function monitored by intraventricular balloon. Cardiac uptake of infused [18F]DHE (50–150 kBq.min−1) was monitored by γ-detection, while ROS generation was invoked by menadione infusion (0, 10, or 50 μm), validated by parallel measures of cardiac oxidative stress. Results [18F]DHE was most sensitive to oxidation by superoxide and hydroxyl radicals. Normalised [18F]DHE uptake was significantly greater in menadione-treated hearts (1.44 ± 0.27) versus control (0.81 ± 0.07) (p < 0.05, n = 4/group), associated with concomitant cardiac contractile dysfunction, glutathione depletion, and PKG1α dimerisation. Conclusion [18F]DHE reports on ROS in a validated model of oxidative stress where perfusion (and tracer delivery) is unlikely to impact its pharmacokinetics.

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